Understanding the mechanisms of an effective neutralizing antibody response to HIV is one of the highest priorities in the field of HIV-specific immunity. In this regard, the inability of the humoral response of most vaccinees to cross-neutralize multiple strains of HIV is believed to be a major obstacle to the design of effective vaccines. In 2006 we observed that sera from subpopulation of our chronically-infected cohorts had considerable neutralization breadth extending across clades. Much of the work done prior to that time had not focused on patients selected for broadly cross-neutralizing antibodies to HIV-1. In addition, a relatively small number of monoclonal neutralizing antibodies existed at the time. For these reasons we began to recruit a cohort of individuals screened for broadly cross-neutralizing antibodies to HIV. We, in collaboration with John Mascola at the Vaccine Research Center (VRC), used these sera to systematically dissect the means by which these patients cross-neutralize. We thus far have identified 30 such patients and are continuing to accrue additional subjects. A number of fundamental questions had not been addressed with regard to the HIV-specific humoral immune response of these patients. For example, it was not known if these patients had genetic or clinical characteristics, or HIV-specific cellular immune response characteristics in common. It was also not known whether neutralization was mediated by a few B cell clones directed to conserved epitopes or by an extremely polyclonal response to many epitopes. Given that our patients are infected with clade B viruses and should not have experienced infection by viruses belonging to multiple clades, we hypothesized that cross-neutralization is mediated through conserved epitopes on HIV envelope. In addition, although considerable work had been done on patient sera, very little had been done on HIV-specific B cells. The phenotype and immunoglobulin class of HIV-specific B cells in comparison to responses to other viruses remained poorly defined. Further, it remained unclear whether patients with broad cross-neutralizing activity are unique with regard to these parameters. One primary objective of our work on the humoral response to HIV is to understand the basis of a broadly cross-neutralizing antibody response in our patients. It was not known whether there are common features of the humoral response of such patients with regard to specificity. It was also not known whether neutralization was mediated by a few B cell clones directed to conserved epitopes or by an extremely polyclonal response to many epitopes. To understand the specificity and diversity of epitopes targeted by the B-cell response in patients with broad sera, we have initiated a collaborative effort to isolate monoclonal antibodies. This work has resulted in several publications using the targeted strategy of isolating monoclonal antibodies from sorted gp140-labelled B-cells. Collaborators at Rockefeller University have observed that gp140-labelled B-cells contain numerous clones with some neutralizing activity that may have an additive effect to produce breadth. However, individual monoclonal antibodies with extraordinary potency and breadth have been isolated in our laboratory or at the VRC. One of these monoclonals, named VRC01, is a very broad and potent antibody that binds the CD4 binding site of the HIV envelope protein. At the time, the isolation of monoclonal antibodies from cells of chronically infected patients was providing considerable advances in understanding the specificities and mechanisms underlying broadly neutralizing antibody responses to HIV-1. However, one limitation to the methods employed is that they used protein probes to isolate B cells which required prior knowledge of the specificity of the antibodies that are being sought. To further understand the specificities and binding characteristics that underlie a broadly neutralizing antibody response we recently developed techniques that permitted isolation of human monoclonal antibodies without previous knowledge of specificity. In this technique peripheral blood memory B cells are sorted and expanded for 13 days with interleukin (IL)-2, IL-21 and CD40-ligand expressing cells. The supernatants of large numbers of micro-cultures of these cells can then be screened for neutralizing activity in a high-throughput manner. From the cultures that exhibit anti-HIV neutralizing activity the immunoglobulin genes can then be isolated, re-expressed, and characterized. In 2013 we isolated an antibody, designated 10E8, that is among the most broad and potent thus far described. It binds the gp41 membrane-proximal external region (MPER) of HIV envelope. It is very potent and neutralizes 98% of tested viruses. An analysis of sera from 78 healthy HIV-1-infected donors demonstrated that 8% contained 10E8-like specificities. In contrast to other neutralizing MPER antibodies, 10E8 did not bind phospholipids, was not autoreactive, and bound cell-surface envelope. The structure of 10E8 in complex with the complete MPER revealed a site of vulnerability comprising a narrow stretch of highly conserved gp41-hydrophobic residues just before the transmembrane region. Analysis of resistant HIV-1 variants confirmed the importance of these residues for neutralization. These data indicate that the highly conserved MPER is a target of potent, non-self-reactive neutralizing antibodies. This suggests that HIV-1 vaccines should aim to induce antibodies to this region of HIV-1 envelope glycoprotein. Through this work we continue to provide a better understanding of the specificities and functions of the HIV-specific humoral immune response that is most likely to provide protection from infection. Over the coming years we anticipate that this work, in the context of work from other groups, will greatly enhance our knowledge of what features of this response should be induced in vaccination strategies.